Microbiological evaluation of the ability of the DEKO-190 Washer/Disinfector to remove Clostridium difficile spores from bedpan surfaces.

BACKGROUND: Clostridium difficile is a major nosocomial pathogen causing mild diarrhoea to life-threatening pseudomembranous colitis, and its spores frequently contaminate hospital environments and equipment. Washer/Disinfectors (WDs) are commonly used to clean and decontaminate soiled equipment in health care facilities. This study aimed to evaluate the effectiveness of the DEKO-190 WD in removing C. difficile spores from bedpans. METHODS: Plastic carriers were inoculated with suspensions of C. difficile spores in autoclaved (sterile) human faeces. The carriers were then taped to a sterile plastic bedpan which was subjected to short, long or intensive wash cycles in the WD using one of two test detergents: Formula A (generic) and Formula B (highly alkaline). Mean log10 reductions in spores were calculated for each wash cycle. RESULTS: Mean log10 reductions were 3.21(SEM ± 0.20) and 2.82 (±0.13) for Formula A and B, respectively, for the short cycle. The mean log10 reductions using the long wash cycle were 3.65 (±0.44) using Formula A and 5.30 (±0.43) using Formula B, while log10 reductions were 3.37 (±0.58) (Formula A) and 4.64 (±0.47) (Formula B) for the intensive cycle. Washing with the DEKO-190 significantly reduced spore concentrations on carrier surfaces on a bedpan. Spore counts were most effectively reduced when carriers were washed on a long or intensive wash cycle using an alkaline detergent.

Comparison of ChromID C. difficile agar and cycloserine-cefoxitin-fructose agar for the recovery of Clostridium difficile.

AIM: The rapidly changing epidemiology of Clostridium difficile infection highlights the need for improved and continuing surveillance involving stool culturing to enable molecular tracking. Culture of C. difficile can be difficult and time consuming. In this report ChromID C. difficile agar (CDIF) was compared to cycloserine-cefoxitin-fructose-egg-yolk agar which contained 0.1% sodium taurocholate (TCCFA) as a germinant. RESULTS: All ribotypes of C. difficile tested (n=90) grew well on CDIF within 24 h and most gave characteristic small irregular black colonies with a raised umbonate profile. Counts from standard suspensions of C. difficile at 24 h (p<0.005) and 48 h (p=0.01) were significantly higher on CDIF than on TCCFA. Similar results were achieved after alcohol shock. When temperature shock was used to differentiate vegetative cells and spores, the total number of culturable and vegetative cells on CDIF was significantly higher than on TCCFA (culturable cells, p=0.003 at 24 h and p=0.002 at 48 h; vegetative cells, p=0.0003 at 24 h and p=0.0002 at 48 h). CONCLUSIONS: These data suggest that CDIF is a better medium for the recovery of vegetative C. difficile than TCCFA and equal to TCCFA for spore recovery.

The effect of an acidic cleanser versus soap on the skin pH and micro-flora of adult patients: a non-randomised two group crossover study in an intensive care unit.

OBJECTIVES: To test the effects of two different cleansing regimens on skin surface pH and micro-flora, in adult patients in the intensive care unit (ICU). RESEARCH METHODOLOGY: Forty-three patients were recruited from a 23-bed tertiary medical/surgical ICU. The nineteen patients in Group One were washed using soap for daily hygiene care over a four week period. In Group 2, 24 patients were washing daily using an acidic liquid cleanser (pH 5.5) over a second four week period. Skin pH measurements and bacterial swabs were sampled daily from each for a maximum of ten days or until discharged from the ICU. MAIN OUTCOME MEASURES: Skin surface pH and quantitative skin cultures (colony forming units). FINDINGS: Skin pH measurements were lower in patients washed with pH 5.5 cleanser than those washed with soap. This was statistically significant for both the forearm (p = 0.0068) and leg (p = 0.0015). The bacterial count was not statistically significantly different between the two groups. Both groups demonstrated that bacterial counts were significantly affected by the length of stay in ICU (p = 0.0032). CONCLUSION: This study demonstrated that the product used in routine skin care significantly affects the skin pH of ICU patients, but not the bacterial colonisation. Bacterial colonisation of the skin increases with length of stay.

Isolation of Clostridium difficile from faecal specimens--a comparison of chromID C. difficile agar and cycloserine-cefoxitin-fructose agar.

The culture of toxigenic Clostridium difficile from stool specimens is still seen as the gold standard for the laboratory diagnosis of C. difficile infection (CDI). bioMérieux have released ChromID Cdiff chromogenic agar (CDIF) for the isolation and identification of C. difficile in 24 h. In this study, we compared CDIF to pre-reduced cycloserine-cefoxitin-fructose agar with sodium taurocholate (TCCFA) in the examination of glutamate dehydrogenase-positive faecal specimens that were either GeneOhm positive or negative, using direct culture or culture following alcohol shock. Direct culture on CDIF had a sensitivity of 100 % and recovery of 94 % while for TCCFA these were 87 % and 82 %, respectively. For GeneOhm-positive alcohol-shocked faecal samples, sensitivity and recovery on CDIF was similar to direct culture while on TCCFA they were about 10 % higher. For direct culture, there was a significant difference between growth on CDIF at 24 h and TCCFA at 48 h (P = 0.001) and between the two media at 48 h (P<0.001). A total of 142 strains of C. difficile were recovered in pure culture from all GeneOhm-positive samples used in this study and 11 (7.7 %) of these were A(-)B(-)CDT(-) and may represent mixed infections of toxigenic and non-toxigenic C. difficile. The most dominant ribotype was UK 014 (14.7 %) followed by 002 (11.9 %) and 020 (11.9 %), and 36 % of toxigenic isolates, including an A(-)B(+)CDT(-) strain, could not be assigned a UK ribotype. CDIF outperformed pre-reduced TCCFA by negating the need for alcohol shock treatment and by giving a time saving of 24 h in the isolation of C. difficile. CDIF plates were also more selective than TCCFA and C. difficile colonies were easy to identify and subculture prior to strain typing.

Candida albicans adhesion to human epithelial cells and polystyrene and formation of biofilm is reduced by sub-inhibitory Melaleuca alternifolia (tea tree) essential oil.

This study investigated the effects of the volatile terpene-rich oil from Melaleuca alternifolia (tea tree oil) on the formation of biofilms and the adhesion of C. albicans cells to both biotic and abiotic surfaces. Biofilm formation on polystyrene was significantly inhibited for 70% of the isolates at the lowest test concentration of 0.016% of tea tree oil (TTO) when quantified by XTT and 40% of isolates when measured by crystal violet staining. Adhesion to polystyrene, quantified by crystal violet staining, was significantly reduced for 3 isolates at 0.031%, 6 isolates at 0.062% and 0.125% and for all 7 isolates at 0.25% TTO. Reductions in adhesion were not due to loss of viability (at concentrations of ≤ 0.125%) or interactions between the TTO and polystyrene. Similarly, adhesion to buccal epithelial and HeLa cells was also significantly reduced in the presence of 0.016-0.062% TTO. Treatment with 0.125% TTO, but not 0.062%, decreased the cell surface hydrophobicity of C. albicans, indicating one potential mechanism by which adhesion may be reduced. These data demonstrate that sub-inhibitory TTO reduces the adhesion of C. albicans to both human cells and polystyrene, inhibits biofilm formation and decreases cell surface hydrophobicity.

Siderophore production by actinomycetes isolates from two soil sites in Western Australia.

The actinomycetes are metabolically flexible soil micro-organisms capable of producing a range of compounds of interest, including siderophores. Siderophore production by actinomycetes sampled from two distinct and separate geographical sites in Western Australia were investigated and found to be generally similar in the total percentage of siderophore producers found. The only notable difference was the proportion of isolates producing catechol siderophores with only 3% found in site 1 (from the north-west of Western Australia and reportedly containing 40% magnetite) and 17% in site 2 (a commercial stone fruit orchard in the hills east of Perth with a soil base ranging from sandy loam to laterite). Further detailed characterization of isolates of interest identified a Streptomyces that produced extracellularly excreted enterobactin, the characteristic Enterobacteriaceae siderophore, and also revealed some of the conditions required for enterobactin production. Carriage of the entF gene, which codes for the synthetase responsible for the final assembly of the tri-cyclic structure of enterobactin, was confirmed by PCR in this isolate. Another separate Streptomyces produced a compound that matched the UV/VIS spectra of heterobactin, a siderophore previously only described in Rhodococcus and Nocardia.

In Vitro susceptibility of methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus to a new antimicrobial, copper silicate.

The soluble copper silicate (CS) MIC of 100 strains of methicillin-resistant Staphylococcus aureus and 100 strains of methicillin-susceptible S. aureus (MSSA) was 175 mg Cu/liter. Bactericidal and postantibiotic effects (> or =1 h) were seen at 2x MIC and 4x MIC. The frequency of mutation was <10(-9), and serial passage could not extend growth beyond 1.6x MIC.

The fhu genes of Rhizobium leguminosarum, specifying siderophore uptake proteins: fhuDCB are adjacent to a pseudogene version of fhuA.

A mutant of Rhizobium leguminosarum was isolated which fails to take up the siderophore vicibactin. The mutation is in a homologue of fhuB, which in Escherichia coli specifies an inner-membrane protein of the ferric hydroxamate uptake system. In Rhizobium, fhuB is in an operon fhuDCB, which specifies the cytoplasmic membrane and periplasmic proteins involved in siderophore uptake. fhuDCB mutants make vicibactin when grown in Fe concentrations that inhibit its production in the wild-type. Nodules on peas induced by fhuDCB mutants were apparently normal in N2 fixation. Transcription of an fhuDCB-lacZ fusion was Fe-regulated, being approximately 10-fold higher in Fe-depleted cells. Downstream of fhuB, in the opposite orientation, is a version of fhuA whose homologues in other bacteria specify hydroxamate outer-membrane receptors. This fhuA gene appears to be a pseudogene with stop codons and undetectable expression.

Rhizobium leguminosarum bv. viciae produces a novel cyclic trihydroxamate siderophore, vicibactin.

Trihydroxamate siderophores were isolated from iron-deficient cultures of three strains of Rhizobium leguminosarum biovar viciae, two from Japan (WSM709, WSM710) and one from the Mediterranean (WU235), and from a Tn5-induced mutant of WSM710 (MNF7101). The first three all produced the same compound (vicibactin), which was uncharged and could be purified by solvent extraction into benzyl alcohol. The gallium and ferric complexes of vicibactin were extractable into benzyl alcohol at pH 5.0, while metal-free vicibactin could be extracted with good yield at pH 8.0. The trihydroxamate from MNF7101 (vicibactin 7101) could not be extracted into benzyl alcohol, but its cationic nature permitted purification by chromatography on Sephadex CM-25 (NH+ 4 form). Relative molecular masses and empirical formulae were obtained from fast-atom-bombardment MS. The structures were derived from one- and two-dimensional 1H and 13C NMR spectroscopy, using DQF-COSY, NOESY, HMQC and HMBC techniques on the compounds dissolved in methanol-d 4 and DMSO-d 6. Vicibactin proves to be a cyclic molecule containing three residues each of (R)-2,5-diamino-N 2-acetyl-N 5-hydroxypentanoic acid (N 2-acetyl-N 5-hydroxy-D-ornithine) and (R)-3-hydroxybutanoic acid, arranged alternately, with alternating ester and peptide bonds. Vicibactin 7101 differed only in lacking the acetyl substitution on the N2 of the N 5-hydroxyornithine, resulting in net positive charge; it was still functional as a siderophore and promoted 55Fe uptake by iron-starved cells of WSM710 in the presence of an excess of phosphate. The rate of vicibactin biosynthesis by iron-deficient cells of WSM710 was essentially constant between pH 5.5 and 7.0, but much decreased at pH 5.0. When iron-starved cultures were supplemented with potential precursors for vicibactin, the rates of its synthesis were consistent with both ß-hydroxybutyrate and ornithine being precursors. At least three genes seem likely to be involved in synthesis of vicibactin from ornithine and ß-hydroxybutyrate: a hydroxylase adding the -OH group to the N5 of ornithine, an acetylase adding the acetyl group to the N2 of ornithine, and a peptide synthetase system.